It is estimated that more than 50% of the United States population has vitamin D (Vit D) insufficiency. Of the tens of thousands of published studies devoted to Vit D, less than a handful have examined Vit D in the cornea and anterior segment and none of these measured Vit D concentrations, metabolism, or activity at those sites. Systemic hypovitaminosis D could lead to or exacerbate anterior segment hypovitaminosis D. This may be of concern following corneal injury given that our preliminary data showing that lack of the vitamin D receptor leads to delayed wound healing. We have also demonstrated that Vit D enhances corneal epithelial barrier function. Apart from its likely immune role in the eye, it has also been suggested that Vit D plays a role in intraocular pressure control and age related macular degeneration. Over the last 2 years we have entered into this new and exciting area of research, namely examining vitamin D (Vit D) in the anterior segment of the eye. The proposed project will continue to lay the foundation for all future Vit D work in te cornea and eye. Our primary hypotheses are that Vit D promotes cell differentiation in the eye, and that Vit D is present throughout the eye and is specifically transported into tear fluid where t can influence epithelial differentiation. The three Specific Aims of this proposal, based on these hypotheses, are (1) Determine if Vit D affects corneal epithelial differentiation; (2) Examine the effects VDR knockout on corneal epithelial wound healing; and (3) Determine how Vit D is transported into the tear fluid. For Aim 1, in-vivo experiments will examine differentiation markers in Vit D Receptor (VDR) knockout mice. In-vitro experiments will examine differentiation markers in cultured corneal epithelial cells exposed to different concentrations of 25(OH)D3 and 1,25(OH)2D3. We will also examine the role of occludin and other Vit D metabolites in corneal epithelial physiology. For Aim 2, corneal epithelial wound healing will be examined in -/- and -/+ VDR knockout mice at different times after birth. Identical groups will be fed a special replenishment diet and corneal epithelial wound healing will be examined. Junctional complexes and differentiation markers will also be examined in all of these mice. In Aim 3, lacrimal fluid wil be collected from the rabbits fed a normal or high Vit D diet and Vit D metabolites will be measured. Vit D binding protein as well as megalin and cubilin (additional significant Vit D protein carriers) will be assayed in all samples to determine possible Vit D transport pathways into the ocular compartments. PUBLIC HEALTH RELEVANCE: It is estimated that more than 50% of the United States population has vitamin D (Vit D) insufficiency (2, 4, 5). Of the tens of thousands of published studies devoted to Vit D, less than a handful have examined Vit D in the cornea and anterior segment and none of these measured Vit D concentrations, metabolism, or activity at those sites. This work will address some of these topics.